Fermentative process for producing ergocryptine



nited States Patent 3,485,722 FERMENTATIVE PROCESS FOR PRODUCING ERGOCRYPTINE Alba Maria Amici, Via Marigi N. 13; Anacleto Minghetti, P.zza Vesuvio N. 23; Tullio Scotti, Via G. del Maino ILS. Cl. 195-81 mineral salts.

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' The new strain Claviceps purpm'ea F.I. 101a has been isolated from ergot sclerotium collected on a rye ear. It has the following morphological characteristics:

MICROSCOPIC ASPECT N. 21; Celestino Spalla, Via L. Soderini N. 21; and 5 L i i T gn lj i Fiardalisi i all f Milan, Italy On the usual solld cultural med1a, the mycehum shows N0 Drawing. Filed July 13, 1967, Ser. No. 654,089 yp which when young are long and thin with hardly Claims priority, application Italy, July 22, 1966, evident and well spaced septa. Upon ageing the hyphae 16,943/66 become larger, the septa become very evident and the C121 1/10; C121? 1/08 10 various cells which constitute the hyphae swell. In young 2 Claims hyphae the length of the cells is 2430,u and the diameter is 34,IL. Old cells are 10-14 long and have a diameter of 4-5 Sometimes the cells which constitute the old ABSTRACT OF THE DISCLOSURE hyphae separate and around their terminal parts give Described is a microbiologic process for the preparation ri e to arthrospores which have dimensions not very diiferof 'yp in Which the microorganism Claviceps ent from the cells. Conidia are formed on top of many Pllrpurea 101a (ATCC 20019) is cultivated in subhyphae more or less quickly and more or less abundantly merged culture under aerobic conditions in a liquid nutridepending on th lt l diu On m dia, th tive medium containing a Source Of carbon, nitrogen, and conidia are roundish and on others clearly val, The

roundish conidia are about 5-7 1 in diameter, and the oval conidia are about 68[L by 35[L.

The present invention relates to a new microbiological M ACROSCOPIC ASPECT method for the preparation of ergocryptme. More particularly, our invention has as its object the microbiologi- Th m r c p ch r ri s h b n ob rved on cal process for producing the ergot alkaloid ergocryptines slants of the media as listed in Table 1, incubated at 28 by mean of a new microorganism Claviceps purpurea C. for 8,16, and 24 days.

F.I. 101a (collection number of the strains of Societa TS medium:-Abundant growth, velvety aspect and Farmaceutici Italia). Claviceps purpzlrea RI. 101a has whi e col r whi h be mes v ol n g g. Th r l been deposited at the American Type Culture Collection mycelium is abundant. The back side of the colony varies (U.S.A.) receiving the index number ATCC 20019 and 30 from colorless to violet. No soluble pigments are formed. at the Commonwealth Mycological Institute (G.B.) re- Abundant conidia.

ceiving the index number IMI 126133. Ergocryptine be- Agar Potato g1uCS63SPaI$e growth, abundant aefial longs to the ergot alkaloids and gives upon hydrolysis mycelium, white, frequently fasciculated as coremi. The lysergic acid, dimethylpyruvic acid, D-proline and 1- back side of the culture is colorless. Soluble pigments are leucine; the molecules thereof being bonded together by absent. Abundant conidia.

amidic bonds. Ergocryptine is usefully employed as hyper- T36 medium:-G0od growth, with large folds, fair tensive and in the therapy of peripheric vascular disturbwhitish aerial mycelium. The back side is colorless; soluble pigments are absent. Numerous conidia.

In literature are known some microbiological processes T31 medium:Wrinkled, bright, whitish, fair growth. for the preparation of alkaloids belonging to the ergot Aerial mycelium absent. Soluble pigments absent. Scarce group (Dutch Patents No. 6,415,127 and 6,405,662; US. conidia.

Patents No. 3,117,917 and 3,110,651 and Japanese Pat- T25S mediumz-Sparse growth, irregular, in little more ent No. 10,250/ 64) by means of which it is possible to or less dome-shaped colonies. Fair aerial mycelium of obtain only mixtures of such alkaloids. We have surprisvelvety aspect and pink color. Violet back side. Soluble ingly found, and this is an object of our invention, that pigments are absent. Abundant conidia. by culturing the new microorganism Claviceps purpurea S.P. medium:-Good growth, spread, slight relief, of PI. 101a it is possible to obtain ergocryptine substantially velvety aspect and whitish color. Back side colorless. free from other alkaloids and that besides remarkable Soluble pigments absent. Conidia absent. advantages in extraction and isolation, we obtain the ergo- T22 medium:Good growth, relief, whitish with colorcryptine in good yields in a highly purified state. less back side. Soluble pigments absent. Conidia absent.

Potato Compounds TS T258 T36 T31 glucose SP T22 Saccharose, g 300 200 300 100 Glucose, g Asparagine, g 10 Meal hydrolysates Cotton seeds, g Peptone, g 1. Yeast extract, g. Citric acid, g

Potato infusion 1 ml Grain chafi infusion, ml

Corn steep, g Potassium chloride, mg Potassium dihydrogen phosphate (KHzPOD, Magnesium sulphate (MgSOh'i H2O), g Ferrous sulphate (FESO4.7 H2O), mg

Zinc sulphate (ZnSO4.7 H20), mg

TO 11 000 ml. 1.. 11 TO 11 1,000 mi.

1 To pH 5.2. 2 200 g. of potatoes are boiled for 45 minutes in 500 ml. tap Water and filtered through gauze. The (infused) filtrate is made up to v ume.

3 200 g. of grain chaff (glumes) are boiled for 45 minutes in 5 liters tap water, filtered and made up to volume.

The process of the invention comprises cultivating the microorganism Claviceps purpurea Fl. 101a in a medium containing sources of carbon, nitrogen and mineral salts and then extracting the alkaloid. In greater detail, the microorganism is developed in a liquid cultural medium under aerobic conditions in submerged culture at from 20 to 30 C., preferably 24 C., for a period from 8 to 16 days. The pH may vary, according to the fermentation medium used, from 4 to 6.5. The carbon source may consist of glucose, saccharose, mannite, sorbite, glycerin, citric acid, succinic acid and other substances in common use. The nitrogen source may consist in ammonia, asparagine, peptone, casein hydrolysates and ammonium salts such as the sulphate and chloride and other substances of common use. Useful mineral salts vary according to the medium used and may comprise chlorides, phosphates, sulphates, magnesium, iron, zinc, manganese, and potassium.

The strain Claviceps purpurea F.I. 101a may be stored by lyophilization, using as suspending agent a liquid which consists of three parts of a 60% saccharose solution and 2 parts of milk. It may subsequently be transferred onto T36 medium (Table 1). The fermentation may be carried out in Erlenmeyer flasks and in laboratory or industrial fermento'rs of various capacity.

The quantity of the alkaloid present in the broth may be qualitatively determined by paper chromatography in comparison with a standard of ergocryptine and quantitatively by a spectrophometric method.

The following examples illustrate the invention.

Example 1 An ergot sclerotium from rye ear was washed thoroughly with tap water, dried and submerged for 2 minutes in a 5% solution of mercuric chloride (HgCI After four successive washings with distilled sterile water, the sclerotium was divided into two parts which were then placed upon two slants of the T36 nutritive medium, having the composition listed in Table 1. The two slants Were kept at 24 C. and then allowed to stand for 20 days. Every 45 days some drops of distilled sterile water were added to each slant so as to maintain the sclerotium fragment at high humidity. After 12 days, upon breaking the surface of the fragment, a tuft of a White mycelium was formed. This tuft was transferred by a handle onto another slant of T36 medium and incubated at 28 C. for 8 days.

The culture so obtained in a test tube was used for inoculating 300 ml. flasks containing 50 ml. of the fol- Distilled water to 1000 ml. pH 5.2, with ammonia. Sterilization: 120 C. for '20 minutes.

The resulting innoculum was incubated for 6 days at 24 C. on a rotary shaker at 220 r.p.m. having a range of 4 cm. The cultures thus obtained consist of a well diffused mycelium, without pellets, and were used to inoculate 300 ml. flasks, each of which contained 45 ml. of the following T25 medium:

Saccharose g 300 Citric acid g 15 Potassium chloride g 0.125 Potassium dihydrogen phosphate (KH PO g 0.5 Magnesium sulphate (MgSO -7H O) g 0.5 Ferrous sulphate (FeSO -7H O) mg 7 Zinc sulphate (ZnSO -7I-I O) mg 6 l- Distilled water to 1000 ml. Yeast extract, 0.1 g. pH 5.2, with ammonia. Sterilization: 100 C. for 20 minutes.

The inoculation quantity used was 5 ml. for each flask.

The resulting innoculum was incubated at 24 C. on a rotary shaker at 220 r.p.m. having a range of 4 cm. After 11-13 days of incubation, the cultures contained a quantity of alkaloid varying between 1200 and 1600 /ml.

The contents of 120 flasks were collected together. The 5 liters of the culture so obtained were filtered. The filtrate and the mycelium were extracted separately. The filtrate was adjusted to pH 9 with sodium carbonate and extracted with 5 liters of chloroform. The chloroform extract is reextr'acted with a 4% aqueous solution of tartaric acid. The tartaric solution was concentrated in vacuo at 2030 C. to A- of the original volume, made alkaline to pH 9 and extracted with chloroform. The mycelium panel was treated with 50% aqueous acetone containing the 4% tartaric acid and filtered. The filtrate was made alkaline to pH 9 and extracted with chloroform. The chloroform extracts of the mycelium and of the filtrate were collected together and evaporated to dryness. 7.2 g. of a crude product so obtained were dissolved in benzene in the ratio of 1:20. The solution was decolorized with Darco carbon G-60, filtered and concentrated in vacuo to about 58 volumes of solvent per gram of alkaloid. After standing long at +5 C., the crystallized ergocryptine was collected and dried in vacuo at 45 C.

The product, crystallized from benzene, was dissolved with stirring in 1.5 volumes of boiling aqueous methyl alcohol. The crystallization began immediately even in the warm and was completed while maintaining the methanolic solution at 03 C. for 4 hous. The crystallized product was collected, washed with a small amount of cold methyl alcohol and dried at C. in vacuo. 5.1 g. of ergocryptine were obtained.

Example 2 The operation was as in Example 1 with the difference that instead of starting from a sclerotium, a culture previously obtained from the sclerotium and stored by subsequent transfers on the T36 medium (Table 1) was used. Furthermore, instead of the T25 medium, the 668 medium was used which has the following composition:

Distilled water to 100 ml. pH 5.2, with ammonia. Sterilization: 100 C. for 20 minutes.

After 14 days of incubation, the cultures contain 1500 7/ cc. of ergocryptine.

Example 3 6 liters of TG medium (Example 1) were sterilized in a 10 liter fermentor by heating at C. for 30 minutes. The medium was inoculated with 50 ml. of a suspension in water of conidia and mycelium obtained. 5 cultures of the strain Clavz'ceps purpurea F.I. 101a were suspended in T36 medium (Table 1) in a test tube. The innoculum were incubated for 4 days at 24 C. with an aeration corresponding to 4 liters per minute and with a stroke of 300 r.p.m. of a rotary shaker having 6 blades. The cultures so obtained served to inoculate, in a ratio of 10%, 6 liters of T25 medium (Example 1) prepared and sterlized in another 10 liter f r entor. The innoculum is incubated at 24 C. with an aeration corresponding to 6 liters of air per minute with a stroke corresponding to 350 rpm. of a shaker provided with 6 blades. After 10 days incubation the culture contained 1100 'y/ml. of ergocryptine.

Example 4 The operation was carried out as in Example 3 with the difference that the process starts from 5 cultures in a test tube on the S.F. cultural medium. 1300 v/ml. of ergocryptine were obtained.

Example 5 Claviceps purpurca E1. 10121 in submerged culture under aerobic conditions in a liquid nutritive medium containing a source of carbon, nitrogen, and mineral salts and thereafter recovering the ergocryptine from the cultured medium.

2. The process of claim 1 which is carried out at from to C. for from 8 to 16 days at a pH between 4 and 6.5.

References Cited UNITED STATES PATENTS ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 

